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M Mackett

Publications and source records attributed to M Mackett.

At least 37 records · Page 2Linked to original sources
PubMed1995

Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.

Animals↗
PubMed1994

Immunization of cottontop tamarins and rabbits with a candidate vaccine against the Epstein-Barr virus based on the major viral envelope glycoprotein gp340 and alum.

The Epstein-Barr virus (EBV) is associated with a range of life-threatening diseases in humans. Development of an effective vaccine has therefore been an important objective. One problem in the development of a subunit vaccine for human administration is the selection of a satisfactory adjuvant since the only one currently licensed for human use is alum, although this is not considered to be very effective. The present study demonstrated that a subunit vaccine composed of the EBV envelope glycoprotein gp340 with alum as the adjuvant did elicit protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins. Furthermore, rabbits immunized with gp340/alum developed the same range of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum. Therefore, these results indicate that alum should be evaluated as an adjuvant as part of a human trial of a gp340-based subunit vaccine.

Adjuvants, Immunologic↗
PubMed1994

Induction of feline immunodeficiency virus-specific cytotoxic T cells in vivo with carrier-free synthetic peptide.

The role of cellular immunity in the establishment and progression of immunosuppressive lentivirus infection remains equivocal. To develop a model system with which these aspects of the host immune response can be studied experimentally, we examined the response of cats to a hybrid peptide containing predicted T-and B-cell epitopes from the gag and env genes of feline immunodeficiency virus (FIV). Cats were immunized with an unmodified 17-residue peptide incorporating residues 196 to 208 (from gag capsid protein p24) and 395 to 398 (from env glycoprotein gp120) of the FIV Glasgow-8 strain by using Quil A as an adjuvant. Virus-specific lymphocytotoxicity was measured by chromium-51 release assays. The target cells were autologous or allogeneic skin fibroblasts either infected with recombinant FIV gag vaccinia virus or pulsed with FIV peptides. Effector cells were either fresh peripheral blood mononuclear cells or T-cell lines stimulated with FIV peptides in vitro. Cytotoxic effector cells from immunized cats lysed autologous, but not allogeneic, target cells when they were either infected with recombinant FIV gag vaccinia virus or pulsed with synthetic peptides comprising residues 196 to 205 or 200 to 208 plus 395. Depletion of CD8+ T cells, from the effector cell population abrogated the lymphocytotoxicity. Immunized cats developed an antibody response to the 17-residue peptide immunogen and to recombinant p24. However, no antibodies which recognized smaller constituent peptides could be detected. This response correlated with peptide-induced T-cell proliferation in vitro. This study demonstrates that cytotoxic T lymphocytes specific for FIV can be induced following immunization with an unmodified short synthetic peptide and defines a system in which the protective or pathological role of such responses can be examined.

Amino Acid Sequence↗
PubMed1993

Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients.

Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins.

Amino Acid Sequence↗
PubMed1993

The Epstein-Barr virus candidate vaccine antigen gp340/220 is highly conserved between virus types A and B.

Anti-Epstein-Barr Virus (EBV) vaccines are being developed which are based on the gp340/220 membrane antigen (MA) gene products from the B95-8 strain. Some proteins are known to be immunologically quite different between type-A (1) and type-B (2) strains of EBV and therefore from a vaccine point of view it was critical to evaluate the degree of conservation of gp340/220. The complete MA coding sequence was determined for two B-type viruses, AG876 and P3HR-1, for comparison with the A-type B95-8. A variable region within MA was sequenced from several other strains. In addition the other open reading frames within the MA-containing BamHI-L fragment of AG876 were sequenced and compared. The results show that there is a high degree of homology between all strains examined. Although some differences were found within the MA coding sequence the only major site of variation was within the repeat region and no consistent A/B changes were found. Monoclonal antibodies generated against A-type MA and representing six epitope groups along the length of the gp340 molecule were found to recognize B-type gp340, thereby demonstrating functional homology. We conclude that, as a vaccine antigen, B95-8 gp340/220 should be equally effective against both types of EBV.

Amino Acid Sequence↗
PubMed1993

MHC class II-restricted presentation of endogenously synthesized antigen: Epstein-Barr virus transformed B cell lines can present the viral glycoprotein gp340 by two distinct pathways.

Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (LCL) efficiently process exogenous antigens for MHC class II-restricted presentation via the chloroquine-sensitive endosomal pathway. Using MHC class II-restricted T cell clones specific for EBV structural proteins, however, we frequently observed significant responses to autologous LCL cells without the addition of exogenous virus. Such responses were reduced by pre-treating the LCL with acyclovir (ACV), a drug blocking productive EBV infection. This suggested T cell recognition of antigen synthesized by LCL cells spontaneously entering virus productive cycle, and led us to question by what route(s) MHC class II-restricted presentation of endogenously synthesized virion proteins was occurring. Cell sorting experiments, using the viral envelope glycoprotein gp340 as a surface marker of productively-infected cells, confirmed that stimulatory activity lay within the gp340-positive fraction. However, closer analysis revealed that most of these cells were not productively-infected but were EBV receptor-positive and had bound released virus. We infer that receptor-mediated delivery of released virus into the endosomal pathway is one route whereby an LCL can present endogenously synthesized EBV proteins on MHC class II molecules. To ask whether another, more direct, route of processing was possible, we used a recombinant vaccinia viral vector to express gp340 de novo in ACV-treated LCLs. Significantly, these cells presented the endogenously synthesized antigen to autologous gp340-specific T cell clones via a chloroquine-resistant pathway. In the same experiments, vaccinia-mediated expression of a signal peptide-deleted form of gp340 did not lead to T cell stimulation, suggesting that this second route of processing required entry of endogenously synthesized antigen into the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of Epstein-Barr virus gp340/220 produced by a bovine papillomavirus virus expression vector system.

Our initial results with a bovine papilloma virus (BPV) vector expression system indicated that we could produce significant amounts of Epstein-Barr virus (EBV) gp340/220 in the supernatant of a mouse fibroblast cell line. We have now extended these findings to show that the truncated version of gp340/220, where the membrane anchor sequence is deleted, is produced even after extended passage of the cells, at a level of approximately 1 mg/4 x 10(8) cells. A simple purification protocol using Sephacryl S300HR and gelatin agarose gives a product which is greater than 90% pure. This product is recognized by anti-gp340 monoclonal antibodies from five different epitope groups and induces antibody that recognizes the authentic gp340/220 and neutralizes EBV in vitro. The purified gp340/220 can be used in ELISA and stimulates the proliferation of T-cell clones specific for gp340/220. These characteristics, together with the fact that BPV-transformed lines have been utilized for the production of pharmaceuticals for use in humans, suggest that this gp340/220 is suitable as a source of antigen for vaccination to prevent EBV infection and related diseases.

Animals↗
PubMed1992

Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector.

Inoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.

Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.

Antibodies, Viral↗
PubMed1991

Diagnosis of nasopharyngeal carcinoma by means of recombinant Epstein-Barr virus proteins.

The immune response of patients with nasopharyngeal carcinoma to Epstein-Barr virus (EBV) antigens is diagnostic of the tumour. Existing tests use EBV antigens produced in EBV-infected lymphoblastoid cells, but the virus replicates poorly in these cells. Serum samples from 18 patients diagnosed as having nasopharyngeal carcinoma were screened by western blot analysis, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence tests for antibodies to the EBV-coded alkaline deoxyribonuclease (DNase), thymidine kinase, and membrane antigen (gp340/220) produced in recombinant baculovirus or bovine papillomavirus systems. Each protein was a useful diagnostic marker for nasopharyngeal carcinoma, although in the gp340/220 ELISAs there was substantial overlap for both IgG and IgA antibodies between serum samples from nasopharyngeal carcinoma patients and those from healthy donors seropositive for EBV. The EBV thymidine kinase was the most sensitive predictor of nasopharyngeal carcinoma; all such samples showed both IgG and IgA antibody responses to this protein and all gave clearly distinct titres from those of the EBV-seropositive donors in the IgA test. Each of the recombinant systems described is suitable for use in large-scale screening programmes for the early diagnosis of nasopharyngeal carcinoma.

Antibodies, Viral↗
PubMed1990

Human cytotoxic T-cell responses against Epstein-Barr virus nuclear antigens demonstrated by using recombinant vaccinia viruses.

The potentially pathogenic effects of infection with Epstein-Barr virus (EBV), a B-lymphotropic agent with cell growth-transforming potential, are contained in healthy virus carriers by virus-specific cytotoxic T-lymphocyte (CTL) surveillance. The target antigens against which such CTL responses are directed are yet undefined, but the antigens probably derived from one or more of the EBV "latent" proteins constitutively expressed in virus-transformed B cells. We have analyzed target specificity of CTL responses from two EBV-immune donors that are preferentially reactive against autologous cells transformed with type A but not with type B virus isolates. Coding sequences for four EBV latent proteins with allelic polymorphism between A and B virus types--namely, the EBV nuclear antigens (EBNAs) EBNA 2, EBNA 3a, EBNA 3c, and EBNA leader protein--have been introduced into vaccinia virus vectors under control of vaccinia promoter P7.5 and used to express relevant EBNA proteins in appropriate target cells. Thus the CTL response from one donor has been mapped to type A EBNA 2 protein and from a second donor to type A EBNA 3a protein. Thereafter, a series of recombinant vaccinia viruses were constructed that carried specific internal deletions within the EBNA 2 type A coding sequence; by using these vectors, the above EBNA 2 type A-specific CTL response was shown to be directed against an epitope within a 100-amino acid fragment near the N terminus of the protein. This work clearly shows human CTL recognition of virus-coded nuclear antigens in the EBV system; moreover, it establishes an experimental approach that can be extended to all EBV latent proteins and to the more common CTL responses that cross-react against type A and type B virus isolates.

Antigens, Viral↗
PubMed1990

Biological characterization of recombinant vaccinia viruses in mice infected by the respiratory route.

A murine model based on infection by the respiratory route has been used to study the pathogenesis of recombinant vaccinia viruses. The neurovirulent Western Reserve (WR) strain and the Wyeth smallpox vaccine strain were used as vectors. Recombinant viruses were constructed by insertion of the Epstein-Barr virus membrane glycoprotein 340 gene into the thymidine kinase (TK) gene of each vaccinia virus. Intranasal inoculation of DBA/2 mice with 10(6) pock-forming units (pk.f.u.) of the WR strain was lethal but mice survived similar infection with the WR recombinant virus. Each virus was recovered from lung, blood and brain but, unlike wild-type virus, the recombinant virus was subsequently cleared. No deaths occurred after similar infection with the Wyeth strain or the Wyeth recombinant virus. There was limited growth of the Wyeth strain in the respiratory tract, low levels of virus in the blood and only sporadic recovery in brain extracts. The Wyeth recombinant virus was cleared rapidly with little viraemia or detectable infection of the central nervous system. No phenotypic character determined in vitro could be related consistently to the virulence of wild-type and recombinant viruses. Although the lethal character of the WR strain was affected by its TK+ phenotype, mice survived infection by intranasal inoculation with 10(6) pk.f.u. of WR TK+ recombinant viruses which either expressed the human interleukin 2 gene or had a deficient vaccinia virus growth factor gene.

Animals↗
PubMed1990

Characterization and expression of a glycoprotein encoded by the Epstein-Barr virus BamHI I fragment.

Computer-assisted analysis of the Epstein-Barr virus (EBV) open reading frame BILF2 (B95-8 nucleotides 150,525 to 149,782) predicts that it codes for a membrane-bound glycoprotein. [3H]glucosamine labeling of cells infected with vaccinia virus recombinants that expressed the BILF2 open reading frame revealed several diffuse species of glycoproteins of around 80,000 and 55,000 daltons. A monoclonal antibody derived from spleens of mice immunized with EBV immunoprecipitated the EBV-derived protein made by the vaccinia virus recombinants and also precipitated a late envelope glycoprotein with a mobility of 78,000 to 55,000 from EBV-producing cells. N-Glycanase treatment of the immunoprecipitated BILF2 product from EBV-producing cells resulted in a polypeptide of 28 kilodaltons, closely agreeing with the predicted molecular mass for the unmodified BILF2 gene product. Western (immuno-) blots using recombinant infected cells as a source of antigen showed that the majority of EBV-seropositive individuals have a serum antibody response to the BILF2-encoded gp78/55.

Amino Acid Sequence↗
PubMed1989

Anti-viral protection and prevention of lymphocytic choriomeningitis or of the local footpad swelling reaction in mice by immunization with vaccinia-recombinant virus expressing LCMV-WE nucleoprotein or glycoprotein.

The viral antigen specificity of primary cytotoxic T cell responses (CTL) of H-2b, H-2k, H-2q, H-2s, H-2f and some H-2-recombinant mice against lymphocytic choriomeningitis virus (LCMV-WE isolate) as well as the specificity of some CTL clones and T cell lines was defined on target cells infected with vaccinia-recombinant virus expressing nucleoprotein (Np) or glycoprotein (Gp). Np was recognized together with H-2q (Dq), H-2d (DLd), H-2s and H-2b (Db). Gp specificity was restricted to H-2f and H-2b (Kb and Db); H-2k-restricted CTL anti-LCMV responses were neither Gp nor Np specific. The anti-viral protective immunity induced by vaccinia-Gp or vaccinia-Np recombinants was evaluated in mice. In vivo protection was T cell mediated by class I restricted Ly-2+ T cells; it correlated well with the CTL specificity defined in vitro. Some of the CTL-nonresponder H-2 allele plus Np or H-2 plus Gp combinations were, however, protected to variable and low degrees by vaccinia-recombinant viruses, indicating that anti-viral protection is a more sensitive readout for CTL activity than the in vitro assay. For example, B10.D2 H-2d mice generated measurable CTL responses only to Np; after immunization with a vaccinia-Np recombinant, LCMV titers were 10(4) times lower in spleens than in vaccinia-primed controls. Although vaccinia-Gp-immunized BALB/c mice revealed no CTL activity in vitro, they nevertheless had 10(2) times lower LCMV titers in spleens than controls. Anti-viral protection, particularly in low-responder combinations, was usually short-lived and diminished after 3 weeks. In a high-responder situation, protection was of a longer duration (greater than 8 weeks). Vaccination with vaccinia-Np or Gp recombinants protected mice against lethal T cell-mediated lymphocytic choriomeningitis induced by LCMV or prevented the local footpad swelling reaction; these in vivo effects were H-2 dependent and followed the identical roles established for CTL recognition in vitro.

Animals↗
PubMed1989

Expression of Epstein-Barr virus membrane antigen gp340/220 in mouse fibroblasts using a bovine papillomavirus vector.

Epstein-Barr virus (EBV) membrane antigen glycoproteins gp340 and gp220 are encoded by a single gene. We have inserted this gene into a bovine papillomavirus (BPV) vector and expressed gp340/220 in mammalian cells under the control of the mouse metallothionein promoter. The proteins produced were of similar Mr, showed similar antigenic specificity and were transported to the same subcellular location as the authentic gp340/220. The inclusion of heavy metal ions in the medium had no effect on the levels of gp340/220, which were approximately the same as those found in standard EBV-transformed lymphoblastoid cell lines, e.g. B95-8. Cells that expressed gp340/220 were selected by several rounds of fluorescence-activated cell sorting, but on passage they rapidly lost the ability to express this glycoprotein. In contrast to this we found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage.

Animals↗